Throughout this application various publications are referred to by partial citations within parenthesis. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the invention pertains.
Neuroregulators comprise a diverse group of natural products that subserve or modulate communication in the nervous system. They include, but are not limited to, neuropeptides, amino acids, biogenic amines, lipids and lipid metabolites, and other metabolic byproducts. Many of these neuroregulator substances interact with specific cell surface receptors which transduce signals from the outside to the inside of the cell. G-protein coupled receptors (GPCRs) represent a major class of cell surface receptors with which many neurotransmitters interact to mediate their effects. GPCRs are characterized by seven membrane-spanning domains and are coupled to their effectors via G-proteins linking receptor activation with intracellular biochemical sequelae such as stimulation of adenylyl cyclase.
Opsins represent one of the major families of GPCRs. These receptors are unique compared to other GPCRs in that light is a crucial co-factor for their activation under physiological conditions. A major subclass of the opsin family is that of visual opsins such as rhodopsin and cone opsins. The visual opsins, also known as visual photopigments, are located in the eye and are involved in transducing visual information from the eye to the brain. Our understanding of opsin function has been derived primarily from the study of visual photopigments.
Rhodopsin and cone opsins are localized in retinal rod and cone photoreceptors, respectively. These photopigments respond to different wavelengths of light and thus have very distinct absorption spectra associated with different absorption maxima (λmax). Even though both receptor subtypes convey visual signals to the brain in response to illumination, they have evolved to perform very distinct functions related to vision. Cone opsins are primarily responsible for color vision, also known as photopic vision, in different species. In contrast, rhodopsin, believed to have evolved from cone opsin, is mainly involved in dim light vision, also known as scotopic vision. Rhodopsin, highly enriched in rod photoreceptor membrane, has been used extensively as a model receptor to understand activation mechanism and functioning of opsins.
Rhodopsin contains the seven membrane-spanning apoprotein opsin and a retinoid-based chromophore (See reviews Hargrave and McDowell, 1992; Yarfitz and Hurley, 1994). In the ground or inactive state (i.e. in the absence of light), the chromophore, usually 11-cis-retinal, is covalently attached to a highly conserved lysine residue in the middle of the seventh transmembrane segment via a protonated Schiff base. All vertebrate visual opsins contain a highly conserved glutamate residue in the transmembrane helix 3 which serves as a counterion for the protonated Schiff base. It has been postulated that 11-cis-retinal behaves as an inverse agonist and induces an inactive conformation of the apoprotein which, by itself, is partially active (Cohen et al., 1993; Surya et al., 1995). Upon absorbing a photon, 11-cis-retinal is isomerized to the agonist all-trans-retinal which introduces distortion in the opsin and initiates a cascade of conformational changes in the molecule. Rhodopsin is first converted to bathorhodopsin, followed by lumirhodopsin, metarhodopsin I and metarhodopsin II states in a sequential manner. Even though most of these transient conformational states are difficult to study biochemically, they can be easily distinguished on the basis of their spectroscopic properties since each state has a unique absorption maximum. Experimental evidence suggests that the formation of metarhodopsin II, a relatively stable state, involves deprotonation of the Schiff base and represents the active conformation of the apoprotein. In this state, the opsin activates the cognate G-protein and initiates the intracellular signaling cascade which ultimately results in transfer of visual information to the brain. Upon hydrolysis of the Schiff base linkage, metharhodopsin II decays into free all-trans-retinal and opsin. All-trans-retinal is transported to the neighbouring retinal pigment epithelial cells where it is converted to 11-cis-retinal via enzymatic reactions. 11-cis-retinal is transported back to retinal photoreceptors where it recombines with the opsin apoprotein to regenerate the rhodopsin molecule.
Even though all visual opsins essentially use the same activation mechanism as rhodopsin, there are some noticeable differences between vertebrate and invertebrate visual opsins (Gartner and Towner, 1995; Yarfitz and Hurley, 1994, Terakita et al., 1998; Arnheiter, 1998). Activation of vertebrate visual pigment results primarily in stimulation of Gt G-protein (also known as transducin) leading to an increase in cGMP phosphodiesterase activity. Initiation of this signaling cascade ultimately results in closure of cation channels and hyperpolarization of the cell. In contrast, opsin visual pigments in invertebrates such as squid and fruitfly activate Gq G-protein and elevate intracellular IP3 and Ca2+ levels (Wood et al., 1989; Nobes et al., 1992; Yarfitz and Hurley, 1994). Another major difference between vertebrate and invertebrate visual opsins is the stability of the active conformation of the receptor. Formation of vertebrate metarhodopsin II, the active conformation of rhodopsin, is rapidly followed by hydrolysis of the Schiff base linkage and dissociation of metarhodopsin II into free all-trans-retinal and opsin apoprotein. It has been suggested that the glutamate counterion in the transmembrane helix 3 aids in the hydrolysis reaction (Gartner and Towner, 1995). In contrast, invertebrate metarhodopsin represents a thermally stable state where the chromophore remains attached to the apoprotein (Kiselev and Subramaniam, 1994). This allows rapid photoisomerization of all-trans-retinal back to 11-cis-retinal within the apoprotein and rapid regeneration or rhodopsin, thus eliminating the need for retinal regenerating tissue (Provencio et al., 1998). The thermally stable metastate of invertebrate photopigment may be formed due to the absence of the glutamate counterion in transmembrane helix 3 of invertebrate visual opsins (Gartner and Towner, 1995).
Most opsins use 11-cis-retinal derived from carotenoids as a chromophore; however, some opsins use 3-hydroxy, 4-hydroxy or 3,4-dehydro isomers of 11-cis-retinal as a chromophore to accommodate the abundant availability of the substituted carotenoids (Gartner and Towner, 1995). Different opsins respond to photons with different wavelengths, a phenomenon known as spectral tuning. Even though the use of a particular retinal derivative as a chromophore contributes to spectral specificity (Gartner and Towner, 1995), the major determinant of spectral tuning is the presence of unique amino acids surrounding the retinal-binding site (Kochendoerfer et al., 1999). For example, substitution of a highly conserved glycine in transmembrane helix 3 of rhodopsin with amino acids of increasing size results in progressive shift of λmax towards the blue wavelength (Han et al., 1996). Similarly, replacement of conserved non-polar residues with hydroxyl amino acids changes the opsin from a green-absorbing molecule to a red-absorbing pigment (Chan et al., 1992).
Even though the visual opsins have been at the forefront of opsin research, scientists are now turning their attention to non-visual opsins (the opsins not involved in transducing visual information) because of their potential involvement in physiological processes such as circadian rhythm and reproduction. The existence of non-visual photopigments in nonmammalian vertebrates was first suggested by Karl von Frisch. He demonstrated that the skin of the European minnow changed color in response to light even in the absence of the eye and pineal gland, and postulated photoreceptive elements in the diencephalon (Foster et al, 1994). Further evidence supporting the presence of non-visual photopigments was obtained in blinded lampreys and ducks which responded to illumination with body movements and gonadal induction, respectively (Foster et al., 1994). Recent studies using histochemical techniques has further corroborated these physiological observations. Silver et al. (1988) immunostained the cerebrospinal fluid (CSF)-contacting neurons with anti-opsin antibody in brains of the ring dove, quail and duck. Similarly, intense immunostaining of the CSF-contacting neurons within the basal brain of the lizard, Anolis carolinensis, was observed with anti-cone opsin antibody (Foster et al., 1993).
Recent molecular cloning of several non-visual opsins is in agreement with the above-mentioned studies. Pinopsin is expressed in the pineal gland of the chicken and is believed to play a role in circadian rhythm (Okano et al., 1994; Max et al., 1995). Interestingly, expression of the pinopsin gene is regulated by light (Takanaka et al., 1998). Max et al. (1998) have demonstrated light-dependent activation of transducin by pinopsin, implying that the pinopsin is a functional photoreceptor. Two other opsins identified in pinealocytes are vertebrate ancient (VA) opsin cloned from the salmon fish (Soni et al., 1997; Soni et al., 1998), and parapinopsin cloned from the channel catfish (Blackshaw and Snyder, 1997). In addition to pineal cells, VA opsin is also localized in the amacrine and horizontal cells of the salmon retina. On the other hand, expression of parapinopsin is confined to the parapineal and pineal organs of the catfish.
Several of the non-visual opsins are, in fact, expressed in the eye. Sun et al. (1997) cloned peropsin from human retina and mouse eye. This opsin is localized exclusively in microvilli of the apical membrane of retinal pigment epithelial (RPE) cells, indicating that it may function as a sensor of retinoids generated in the adjacent outer membrane of rhodopsin or cone opsins. RPE retinal G-protein-coupled receptor (RGR) is another receptor found in the RPE (Tao et al., 1998). Unlike other opsins which are believed to be present at the plasma membrane, RGR is localized intracellularly. The amino acid sequence of RGR suggests that, along with squid retinochrome, it may form a distinct subfamily of opsins (Hara-Nishimura et al., 1990).
Interestingly, it has been suggested that RGR may prefer all-trans-retinal, rather than 11-cis-retinal, as a ligand and may be involved in the photoisomerization of the all-trans isomer to the 11-cis isomer (Hao and Fong, 1999). In such a case, its function may be the rapid regeneration of 11-cis-retinal in the RPE for use in the visual cycle.
One of the known photo-sensitive processes is melanosome dispersion in the dermal melanophores of Xenopus laevis. In accordance with this, melanopsin has been cloned from melanophores (Provencio et al., 1998). Melanopsin is expressed in the melanophores, suprachiasmatic and preoptic nuclei of the hypothalamus, iris, RPE and retina. Its expression in visual and nonvisual tissues suggests a role in visual and nonvisual photosensory phenomena. Recently, a non-visual opsin has been identified in the mammalian brain. Blackshaw and Snyder (1999) have cloned encephalopsin, which, as the name suggest, is highly expressed in various areas of the brain. It is present in the preoptic area and the paraventricular nucleus of the hypothalamus, the cerebral cortex, cerebellar Purkinje cells, striatum, thalamus and the ventral horn of the spinal cord. Interestingly, this receptor is not present in the eye.
The molecular identified of non-visual opsins has raised several questions. How are they activated? What is their physiological function? All the non-visual opsins cloned to date contain lysine in the seventh transmembrane helix, the site for retinal chromophore attachment, implying that a retinoid may be the chromophore for the non-visual opsins, similar to the visual opsins. Several groups have been successful in reconstituting non-visual opsins with retinoids and activating them with light (Okano et al., 1994; Soni et al., 1998). Retinoids can cross the blood-brain barrier, albeit at low efficiency (Pardridge et al., 1985; Franke et al., 1999). Furthermore, a transporter with high affinity for retinoids, β-Trace, has been recognized recently (Tanaka et al., 1997). This secretory protein is present in high levels in the CSF and may transport retinoids to different regions of the brain, analogous to the plasma RBP. That retinoids are indeed present in the brain was demonstrated by Foster et al. (1993) who were able to identify retinal isomers in the Anolis anterior brain using HPLC analysis. If a retinal isomer is indeed a chromophore for non-visual opsins then light would be needed to photoisomerize the isomer and activate the receptor. Several reports suggest that light can reach the deep areas of the brain (Muller and Wilson, 1986; Grace et al., 1996; Blackshaw and Snyder, 1999), and a neurotransmitter release-enhancing effect of light on cortical slices has been observed (Wade et al., 1988). Therefore, the activation mechanisms of non-visual opsins may be similar to the visual opsins. However, it should be noted here that some non-visual opsins have proven resistant to functional reconstitution with retinal isomers (Provencio et al., 1998; Blackshaw and Snyder, 1999), raising the possibility that these receptors utilize a non-retinoid ligand and may not require light for activation.
What could be the function of non-visual opsins? One interesting possibility is that they may be involved in circadian rhythm. Circadian rhythm represents daily fluctuations in biological activities that are regulated by the light-dark cycles. It is composed of three components: a photoactive input, the circadian clock itself which exhibits periodicity, and the behavioral and physiological oscillations as output. In animals, in the phenomenon known as photoentrainment, exposure to light results in regulation of the circadian rhythm. However, the identity of the photoreceptive molecule mediating photoentrainment has remained a mystery. Since the photoentrainment response occurs at the wavelength of 500 nm, it has been suggested that an opsin may be mediating the response (Foster, 1998).
Freedman et al. (1999) and Lucas et al. (1999) recently demonstrated that photoentrainment was intact in mice lacking rod and cone receptors; however, removal of the eyes in these mice abolished the effect of light on circadian rhythm as well as on melatonin synthesis. These results, and ocular localization of several non-visual opsins, strongly support the role of ocular non-visual opsins in photoentraining circadian rhythm. In addition, the non-visual opsins localized in the CNS may form a component of the circadian clock itself.
If non-visual opsins are indeed involved in regulating circadian rhythm, then they represent an attractive therapeutic target for circadian rhythm-related conditions. These include sleep disorders such as jet lag. It has been suggested that a change in circadian rhythm may be an underlying cause for sleep disorders such as insomnia, Advanced Sleep Phase Syndrome and Delayed Sleep Phase Syndrome (Sedgwick, 1998; Refinetti, 1999). In addition, dissociation between biological clock and work hours may result in shift-work-related sleep disorders. Importantly, bright light therapy has been demonstrated to help in these disorders (Rosenthal et al., 1990; Lack and Wright, 1993; Campbell et al., 1995; Murphy and Campbell, 1996; Cooke et al., 1998; Refinetti, 1999). Similarly, exposure to light at appropriate time reduces the effect of jet lag on travelers (Refinetti, 1999). These observations suggest that non-visual opsins may mediate these beneficial effects of light in circadian rhythm-related disorders.
Non-visual opsins also may play a role in seasonal affective disorder (SAD). This disorder is characterized by a subpopulation of people suffering from depression during winter. Light therapy is effective in these people (Terman and Terman, 1999); especially green light is more effective than red light (Oren et al., 1991). It has been recently hypothesized that the interaction of specialized photoreceptors with magnetic field may influence sensitivity of patients suffering from SAD to light (Partonen, 1998).
In addition, the discreet localization of various opsins in the CNS areas indicate their potential role in CNS-related physiology and disorders.
Non-visual photoreceptors are also involved in melanosome dispersion in melanophores, and thus, in change in the color of the skin in various species. In birds and mammals, non-visual ‘deep brain photoreceptors’ are also linked to reproductive behaviour and photoperiodic gonadal responses (Yoshikawa and Oishi, 1998).
In summary, opsins constitute an important branch of the GPCR superfamily. They behave as photosensitive elements. They are localized in the retina and in non-retinal locations including the brain. The retinal rod and cone opsins are mainly responsible for conveying visual information to the brain, while the non-visual opsins in the retina and elsewhere may be involved in regulation of melatonin synthesis and circadian rhythm, photoentrainment, SAD, skin colour change and camouflage, and reproductive behaviour.